SA^.3 


Reprinted  from 

The  Journal  of  Infectious  Diseases,  1907,  Supplement  No,  3,  pp.  41-49 


SANITARY  CHEMICAL  EXAMINATION  OF  WATER 
BACTERIA/ 

Andrew  Watson  Sellards. 

(State  Water  Survey,  University  of  Illinois,  Urbana,  Illinois.) 

In  view  of  the  modern  theory  of  disease  the  problem  of  sanitary 
water  analysis  concentrates  itself  on  the  bacteriological  rather  than 
on  the  chemical  condition  of  the  water  in  question,  whatever  the 
method  may  be  of  arriving  at  this  knowledge.  All  waters  would  be 
perfectly  safe  for  drinking  purposes,  with  certain  well-characterized 
exceptions  in  the  case  of  mineral  constituents,  if  only  the  pathogenic 
bacteria  were  removed.  Our  investigations  are,  therefore,  directed 
toward  those  bacteria  that  are  pathogenic  to  man,  are  capable  of 
being  borne  by  water,  and  have  the  possibility  of  infecting  the  human 
subject  through  the  alimentary  tract. 

The  routine  sanitary  chemical  tests,  all  of  which  are  for  substances 
perfectly  harmless  in  themselves,  are  merely  an  indirect  method  of 
determining  the  bacteriological  condition  of  the  water.  Naturally 
with  such  an  indirect  method  there  is  a very  wide  range  of  possibilities 
in  the  interpretation  of  the  results.  The  analytical  data  of  a sanitary 
chemical  analysis  indicate  merely  the  presence,  recent  presence,  or 
possible  future  presence  of  pathogenic  bacteria.  It  follows  accord- 
ingly that  by  current  methods  the  sanitary  chemical  analysis  of  pure 
distilled  water  to  which  a little  sterile  bouillon  had  been  added  would 
indicate  a dangerously  polluted  water. 

Naturally,  with  the  advancement  of  bacteriology,  more  direct 
methods  of  analysis  have  been  undertaken.  These  are  of  course 
limited  to  quantitative  and  qualitative  investigations  of  the  bacteria. 
One  does  not  need  to  emphasize  the  difficulty  of  establishing  a stand- 
ard for  the  maximum  number  of  bacteria  permissible  in  a potable 
water,  or  of  the  uselessness  of  searching  the  Mississippi  River  or  the 
Great  Lakes  for  a typhoid  bacillus. 

> This  work  was  carried  on  under  co-operative  agreement  between  the  Illinois  State  Water  Survey, 
State  Geological  Survey,  Engineering  Experiment  Station  of  the  University  of  Illinois,  and  the  Division 
of  Hydro-Economics  of  the  United  States  Geological  Survey. 

41 


19o!0 


42 


Andrew  Watson  Sellards 


The  only  qualitative  work  attempted  at  present  is  the  identification 
of  intestinal  forms.  The  presumptive  coli  tests  are  in  a very  uncertain 
and  unsatisfactory  condition.  The  quantitative  estimation  of  coli 
is  well-nigh  impossible  in  a laboratory  where  a large  number  of  sam- 
ples are  received  for  daily  routine  examination.  Though  possible 
presence  or  absence  can  be  quite  satisfactorily  made  out,  even  when 
many  samples  require  attention  at  the  same  time,  an  opinion  cannot 
be  based  on  a qualitative  test  alone.  It  is  essential  to  know  the 
quantity  of  coli  bacteria  present. 

Of  the  various  routine  methods  of  procedure,  the  direct  chemical 
analysis,  being  the  broadest  of  all,  has  one  advantage  over  all,  or  one 
disadvantage  as  the  case  may  be.  Such  an  analysis  does  not  depend 
on  the  presence  of  living  active  organisms  as  do  all  bacterial  methods ; 
but  the  pollution  may  still  be  detected  where  bacteria  have  died  in 
large  quantities  after  exhausting  their  food  material.  Also  the 
chemical  analysis  will  detect  the  pollution  where  soil-filtered  sewage 
reaches  a water-supply.  Probably  the  most  necessary  factor  in  inter- 
preting the  sanitary  chemical  analysis  is  a thorough  knowledge  of  the 
source  of  the  water  under  examination.  In  this  section  of  Illinois 
one  is  utterly  helpless  in  the  interpretation  of  the  analysis  of  a well 
water,  unless  he  knows  whether  the  well  is  deep  or  shallow  and  in 
drift  or  in  rock. 

The  results  obtained  by  analyzing  the  water  from  two  wells  in  Urbana  will  illus- 
trate the  difficulty.  Amounts  are  stated  in  parts  per  million. 


No.  I 

No.  2 

Total  residue  on  evaporation  . 

• 415- 

365- 

Chlorine  in  chlorides  .... 

3-5 

7-5 

Oxygen  consumed  .... 

5.2 

2 .0 

Nitrogen  as  free  ammonia 

3.6 

0.054 

Nitrogen  as  albuminoid  ammonia  . 

0.136 

0.056 

Nitrogen  as  nitrites  .... 

0 . 040 

0.030 

Alkalinity 

• 353- 

245- 

I c.c. 

1 c.c. 

Bacteria  at  20°  C 

200  . 

1000 . 

B.  coli  communis  .... 

Absent 

Present 

No.  I is  from  a well  i8o  feet  deep  and  has  high  oxygen  consumed  and  high  free 
and  albuminoid  ammonia,  which  are  characteristic  of  deep  wells  in  the  drift  in  central 
Illinois.  Bacterial  tests  show  that  the  water  is  in  good  condition. 

No.  2 is  a water  from  a 30-foot  dug  well,  and  chemically,  according  to  usual  methods 
of  interpretation,  it  is  a better  water  than  No.  i.  Bacterially  it  contains  1,000  bacteria 
per  C.C.,  and  the  reaction  is  positive  to  the  presumptive  test  for  coli. 

Generally  speaking,  the  chemist  must  know  the  normal  constituents  of  a given 
locality  before  he  can  determine  the  amount  due  to  pollution. 


Chemical  Examination  of  Water  Bacteria  43 

In  addition  to  the  sanitary  chemical  analysis  and  the  quantita- 
tive and  qualitative  bacteriological  examination,  a further  hne  of 
procedure  is  suggested  to  the  sci^tist,  that  is,  the  chemical  analysis 
of  bacterial  cultures  obtained  by  the  inoculation  of  water  samples 
into  artificial  media. 

Our  experiments  upon  this  principle  are  based  on  the  supposition 
that,  by  the  inoculation  of  water  into  artificial  sterile  media,  we  could, 
in  a way,  imitate  the  changes  that  would  ordinarily  be  brought  about 
by  bacteria  in  a water  containing  natural  media.  One  great  advan- 
tage would  lie  in  the  ease  with  which  the  artificial  media  could  be 
analyzed,  and  the  accuracy  with  which  the  changes  due  to  the  water 
could  be  determined.  By  this  means  it  is  possible  that  local  factors 
which  affect  the  interpretation  of  the  results  of  analysis  may  be 
removed.  There  arises  the  possibility  of  establishing  an  absolute 
standard  for  the  maximum  limits  of  impurities. 

To  establish  the  value  from  the  sanitary  standpoint  of  the  analysis 
of  the  cultures  of  water,  our  experiments  are  carried  out  with  a view 
to  securing  constancy  of  results.  After  a few  preliminary  tests  (see 
Table  i)  we  chose  for  a medium  an  ordinary  meat-extract  broth  of 
double  concentration,  to  which  2 per  cent  of  gelatin  was  added. 
The  presence  of  sugars  prevented  decomposition  of  the  nitrogenous 
constituents.  When  it  was  desired  to  study  the  nitrogenous  decom- 
position products,  sugars  were  not  added  to  the  medium,  although 
the  extremely  small  amount  of  sugar  present  in  ordinary  acid  meat 
extract  was  not  removed. 

For  our  first  experiments  we  chose  two  types  of  water,  one  from  a 
small  creek,  known  to  be  seriously  polluted,  the  other  from  deep,  driven 
wells  of  unquestionable  purity.  We  measured  accurately  5 c.c.  of 
each  medium  into  test-tubes,  inoculated  with  i c.c.  of  each  sample 
of  water,  and  incubated  at  37°  C.  In  these  preliminary  tests,  the 
cultures,  when  they  showed  appreciable  differences,  were  steamed  in 
an  Arnold  sterilizer  20  minutes,  diluted  to  1,000  c.c.,  and  subjected 
to  sanitary  analysis.  A priori  it  was  expected  that  it  would  be  neces- 
sary to  work  with  very  young  cultures,  presuming  that  for  old  cul- 
tures the  ultimate  analysis  of  a pure  and  of  a polluted  water  would 
be  very  similar.  The  waters  tested  thus  far  have  given  decidedly 
the  contrary  result.  Table  i is  characteristic  of  the  effect  of 


44 


Andrew  Watson  Sellards 


variations  in  media  and  age  of  culture.  The  results  are  expressed 
in  parts  per  million  of  the  diluted  culture,  5 c.c.  of  the  inoculated 
medium  diluted  to  1,000  c.c.  with  pure  distilled  water. 

TABLE  I. 

Comparisons  of  the  Action  of  Pure  and  Polluted  Water  on  Media  of  Various  Com- 
positions FOR  Different  Periods  of  Time. 


Sample 

Oxygen 

Consumed 

Acidity 

Ammonia 

Total 

Solids 

Nitrates 

Chlo- 

rides 

Media 

Free 

Albuminoid 

Hours. . . 

24 

89 

24 

89 

24 

89 

24 

89 

24 

89 

24 

89 

89 

hrs. 

hrs. 

hrs. 

hrs. 

hrs. 

hrs. 

hrs. 

hrs. 

hrs.  hrs. 

hrs. 

hrs. 

hrs. 

Tap  .... 

102 

75 

12.13 

12.13 

0.64 

1. 12 

8.00 

13-6 

212 

0.48 

0.40 

37-0 

Creek... . 

90 

55 

12.13 

21.82 

4.60 

13-20 

8.00 

4.8 

124 

0.40 

0.44 

35-5 

Broth 

Control . . 

75 

7.27 

0.64 

6.80 

254  ■ 

0.48 

37-5 

Tap 

165 

170 

16.97 

15-52 

0.  72 

1 . 00 

8.80 

14. 

386 

0.40 

36-0 

Creek  — 

145 

145 

36.37 

54-80 

0. 76 

1 .00 

7.60 

12.0 

309 

0. 72 

36-0 

Glucose 

Control. . 

165 

14-55 

0.64 

6.4 

433 

0.52 

35-0 

Broth 

Tap 

94 

92 

14-55 

19.40 

0.52 

1.04 

18.80 

23-2 

428 

308 

■ 56 

34-5 

Creek... . 

104 

60 

24-25 

67.90 

3-6o 

23.20 

18.80 

10.4 

397 

168 

0.64 

32.5 

Gelatin 

Control. . 

85 

9.70 

0.64 

13-2 

359 

0.52 

32-5 

Broth 

Tap 

i8s 

183 

21.82 

26.67 

0. 76 

1 . 12 

18.00 

23.20 

612 

489 

0.72 

0.48 

36.5 

Gelatin 

Creek .... 

195 

163 

33-95 

60.62 

0.84 

1.36 

19. 20 

22.4 

574 

400 

0.80 

0.60 

36.0 

Glucose 

Control. . 

180 

16.97 

0.64 

13-60 

557 

0.44 

37-0 

Broth 

As  the  most  striking  changes  took  place  in  the  nitrogenous 
constituents,  we  decided  to  secure  a series  of  preliminary  tests  deter- 
mining only  free  ammonia.  We  were  sometimes  able  to  nesslerize 
directly,  though  more  frequently,  on  direct  nesslerization,  we  secured 
a greenish  color,  not  comparable  with  a true  nessler  color. 

In  an  attempt  to  establish  a maximum  limit  of  free  ammonia, 
allowable,  we  obtained  samples  from  deep  wells  of  unquestionable 
purity,  from  the  best  available  shallow  wells,  and  from  chemically 
pure  water  artificially  polluted.  In  the  following  table  the  cultures 
were  analyzed  at  different  ages  as  a sufficient  variety  of  waters  had 
not  yet  been  analyzed  to  determine  the  most  favorable  age  of  culture. 

In  Table  2 the  following  facts  are  of  special  interest.  The 
previous  statement  that  the  chemical  analysis  of  pure,  deep,  and 
shallow  ground  waters  is  entirely  different  is  verified.  Noticeable 
examples  are  Nos.  18  and  19.  In  No.  18  and  No.  19  the  results 
of  the  bacteriological  analysis  are  practically  the  same  and  also  the 
chemical  analysis  of  the  cultures  of  the  two  agree  very  well.  This 
strengthens  our  hope  that  a universal  standard  of  purity  may  be 
established. 


Comparison  of  Sanitary  Analyses  of  Waters,  from  Various  Sources,  with  the  Ammonia  that  is  Obtained  from  Media  by  Inoculation 


Chemical  Examination  of  Water  Bacteria 


45 


Analysis  of  Culture, 

P.  M.  1-200  Dil. 

Age  Cult. 

in  hrs. 

Tj-  Tf  . vo  0"0  O'  - 0000  - irifo  -ooooococoooooooooooo  -oooo 

<S<N  .HVOI-IVO  •'O'O  •'O'O 

Ammonia 

Alb. 

••••O'OM  

. . • . Ci  . XTisQ 

• • . . M • . . • 

Free 

'000c^'00<000'0’*t  'OnOOOOOCOCO  ‘WO 

COCO  O H lo  lo  0 M O 

OiOMOOHOOMOvMO’^OHMcioOOOOOOOv  .mO 

H CJ 

Chemical  Analysis,  Parts  per  Million 

Chlo- 

rides 

loo  lOO  0 lOlOlOlO00lO>OIO0  loo 

••  ••  • ooo 

fOVO  fO'O  • • 

Alka- 

linity 

r'CO'O  itt^MINOO  rj-... 

co^  (Ti  TtvO  M H O M tT  ... 

lor^  COM  o ... 

POPO  WW  fO  N(SCOWPOWWP<)POPO  ... 

Oxygen 

Con- 

sumed 

m*  lolo  • loo^ooO'oioio 

Mvo  • wot^Oiot^r^'^sOt^  O'Cco 

lOO  • MNN  mmmvomO  <nOO 

Total 

Solids 

»OlO  004  00  WIO^^O'OHOCJIO  0** 

M C4  lo  cs  0 t^'O  OO  CO  C4  PO  <N  O • • 

^PO  POPOO  lOPOPOPOiO^iO^POPO  iO»* 

Nitrogen  as 

Nitrates 

lO  OOOOOOO  too 

Tt-oo  o w C4  rr  o\  ■^00  ^-•o 

0^0  TJ-PO  PO  tOMOOOO^OPOto  ooo 

do  do  d oood-o  ooo  loo 

Nitrites 

8h  ? 2?>8J?8  8 8 8 8;?  S88 

OO  OO  O OOOHOOOOOO  ooo 

d d d d d ’ ' d ’ d 

Albumi- 

noid 

0.136 

0.64 

0.336 
0. 144 

O.OQ4 

0.048 
.056 
.016 
0. 128 
0.64 
.056 
.082 
0.184 
0.094 
0.64 

0.05 

0.016 

0.010 

Free 

0 "T  0 't  ’too  0 N N 00  w 00 

W M PI  M lO'C  '^00  <000  0 <0  PI  CO  W 

lOOO  MO  0 OOt^PlOOO'OOOO  ooo 

fo  PI  dd  0/-^  0 MO  pooci  ooo 

CJ  0 0 

Bacterial 

Analysis 

Coli 

in 

one 

c.c. 

-f 

(abov 

(abov 

(abov 

(abov 

-f 

?-f 

+ 

(abov 

+ 

+ 

+ 

+ 

No,  per 
c.c. 

00  . . , .OOOMO  . OOOOOOVPOIOPSO  ooo 

00  oOOOChO<NOOo  00<N<Nhhpo  tosO  O O 0 to 

Mcncftcoe/iM  h(/3  to 

cd  ci 

Sample 

Lab. 

No. 

m 4;  <u  0 <u 

CCCfl  lOTj-  t^p  OOO'OM  TJ--0  r-00  0 PI 

S959  pifo  piS  t"  t^oo  oo  00  00  00  00  O'  O' 

<»  f o O'  O'  O'  O'  O'  O.  O'  O'  O'  O' 

C/3  c/:  CO  C/3  ^ rfVJ 

Source 

Tap,  deep,  driven  wells 

“Boneyard”  polluted  creek 

Control 

Tap,  deep,  driven  wells 

Driven  wells 

“Boneyard” 

“Boneyard” 

Control 

Vermillion  River,  raw 

Vermillion  River,  filtered 

Control 

Country  well,  30  feet  deep 

Tap,  deep,  driven  weUs 

Control 

Shallow  well,  20  feet 

Shallow  well,  30  feet 

Deep  well,  driven 

Shallow  well,  30  feet 

Shallow  well,  40  feet 

Driven  well 

Driven  well,  30  feet 

Driven  well,  125  feet 

Shallow  well,  18  feet 

“Bonevard”  polluted  creek 

Maximum  for  this  locality  for 

shallow  well 

Sewage  i-i  ,000  dilution 

Sewage  1-10,000  dilution 

No. 

MW  CO  •'t  loio  t~00  O'  0 M w CO  'T  iO\0  t'OO  00  MW 

46 


Andrew  Watson  Sellards 


Analyses  No.  7 and  No.  8,  raw  and  filtered  river  waters,  furnish 
another  good  example  to  support  our  theory.  On  the  whole,  direct 
chemical  analyses  do  not  show  the  true  efficiency  of  a water-filtration 
plant,  as  the  results  approximate  each  other  rather  closely.  The 
bacteriological  analyses  show  decided  differences,  about  97  per  cent 
of  the  bacteria  having  been  removed  by  filtration.  The  chemical 
condition  of  the  two  cultures  varies  widely.  Ordinarily  this  would 
be  adding  comparatively  Httle  to  the  results  of  colony  counts  which 
show  the  bacterial  efficiency  of  the  filter;  but  in  this  case,  although 
the  efficiency  of  the  filter  was  95.4  per  cent  (there  being  only  120 
bacteria  per  c.c.  in  the  filtered  product)  the  water  responded  to 
presumptive  coh  tests.  After  a long  series  of  cultures  no  typical  coli 
communis  could  be  isolated,  while  the  culture  analysis  was  obtained 
on  the  third  day  with  comparatively  little  effort.  Even  this  time  was 
unnecessarily  long.  It  seems  to  us  that  the  relation  of  this  test  to  the 
presumptive  coli  tests  will  bear  further  investigation. 

The  analysis  of  cultures  may  also  show  qualitative  as  well  as 
quantitative  differences  in  water  as  indicated  in  samples  No.  9 and 
No.  10.  Here  the  chemical  analyses  are  comparable  and  the  number 
of  bacteria  practically  the  same.  There  is  a difference  in  the  quality 
of  the  bacteria  as  shown  by  the  presumptive  coli  test  and  this  differ- 
ence shows  up  distinctly  upon  analyzing  the  culture.  No.  9,  which 
gave  the  positive  coli  test,  gave  4.8  parts  of  free  ammonia  to  the 
culture,  while  No.  10,  which  gave  the  negative  test  for  coli,  gave  only 
o . 2 parts  of  free  ammonia  to  the  culture. 

Realizing  the  importance  of  nitrite  determinations  in  water  analysis, 
we  undertook  to  study  their  action.  As  indicated  in  our  first  table, 
the  action  of  nitrites  and  nitrates  could  best  be  studied  in  media  to 
which  nitrites  and  nitrates  had  been  added.  We  accordingly  added 
0.05  per  cent  sodium  nitrite  (NaNOa)  to  part  of  our  ordinary  media. 
The  results  of  a single  experiment  were  as  follows : 

Nitrite  Media  Cultures. 

Tap  water  gave  . . . . 7.5  parts  per  million  N as  nitrite. 

Boneyard  gave  ....  0.0  “ “ “ “ “ “ 

Media  Control  gave  ...  7.5  “ “ “ “ “ “ 

The  above  test,  we  believe,  differentiated  very  sharply  in  this 
instance,  between  the  nitrites  formed  by  putrefactive  bacteria  and  the 
nitrites  normally  very  high  in  a pure  water. 


Chemical  Examination  of  Water  Bacteria 


47 


According  to  present  methods  of  interpretation  a good  water  may 
be  condemned  on  high  nitrites  alone;  but  the  absence  of  nitrites 
does  not  show  that  a water  is  good.  Further,  the  presence  of  nitrites 
in  shallow  wells  is  assumed  to  be  due  not  to  normal  constituents  of  the 
soil,  but  to  bacterial  action.  It  is  still  further  assumed  that  the  bac- 
teria concerned  are  putrefactive  forms.  Possibly  the  extent  of  the 
assumption  is  not  thoroughly  understood.  Deep  ground  waters 
often  contain  high  nitrites,  their  presence  being  explained  by  the 
supposition  of  the  reduction  of  the  nitrates  present  by  the  ferrous  iron. 
We  have,  however,  isolated  very  active  denitrifying  bacteria  from 
these  waters,  and  also  from  the  air.  Such  bacteria  might  very 
naturally  gain  access  to  shallow  wells  containing  nitrates  and  form 
nitrites.  As  compared  with  the  relatively  few  denitrifying  bacteria, 
there  is  the  great  mass  of  nitrifying  bacteria  universally  present  in  the 
soil  and  upon  which  the  preservation  of  life  upon  the  face  of  the  earth 
depends.  When  we  consider  these  two  great  classes  of  bacteria  it 
does  not  seem  to  us  to  be  proven  that  the  presence  of  nitrites  should 
condemn  a surface  water. 

The  sanitary  chemical  tests,  developed  as  they  were  on  an  entirely 
empirical  basis,  before  the  science  of  bacteriology  was  scarcely  begun, 
were  naturally  difficult  of  interpretation  and  mistakes  were  unavoid- 
able.^ Our  experiments  indicate  that  the  present  interpretation  of 
nitrites  is  partly  in  error,  in  that  high  nitrites  could  not  normally  be 
present  in  a shallow  well. 

Except  in  extreme  cases  it  is  often  impossible  to  give  an  opinion  of 
an  Illinois  water  from  the  ordinary  sanitary  data,  chemical  and  bac- 
teriological, simply  on  account  of  the  lack  of  evidence  furnished 
concerning  the  source  of  the  sample.  Other  authors  have  had  the 
same  experience.  M.  O.  Leighton^  says: 

There  has  been  in  the  past  surprisingly  little  discrimination  used  with  reference 

to  the  selection  of  determinations  for  specific  purposes If  ....  it  is 

desired  to  determine  the  amount  of  organic  pollution  in  a water  and  show  its  value  for 
domestic  use,  the  chemist  forthwith  begins  his  round  of  nitrogen  determinations,  and 
closes  with  a statement  of  the  oxygen  consumed  and  the  number  of  bacteria  per  c.c. 
In  only  a few  well-known  laboratories  has  this  rule  been  violated,  and  such  is  the 
conservatism  in  the  chemical  profession  that  it  will  probably  be  largely  followed  in 

* W.  P.  Mason,  Examination  of  Water,  2d  ed.,  New  York;  criticism  of  Wanklyn. 

* T.  M.  Prudden,  Drinking-Water  and  Ice  Supplies,  2d  ed.,  New  York,  igoi. 

Field  Assay  of  Water,  Water  Supply  and  Irrigation  Paper,  No.  151,  pp.  10  and  ii. 


48 


Andrew  Watson  Sellards 


the  future The  occasional  isolated  sanitary  analysis  of  a water  is  positively 

without  value.  There  are  throughout  the  country  numerous  state,  municipal,  and 
private  laboratories  in  which  sanitary  analyses  are  carried  on.  The  water  analyzed 
may  be  today  from  a well,  tomorrow  from  a brook,  and  the  next  day  from  a pond. 
From  the  results  of  a single  analysis  wise  and  ponderous  verdicts  are  sent  broadcast, 
and  the  eager,  waiting  public  is  duly  impressed. 

As  the  basis  of  our  experiments  we  have  taken  up  the  interpre- 
tation of  chemical  data  from  a theoretical  standpoint,  and  we  believe 
that  this  standpoint  is  absolutely  essential  to  the  intelligent  under- 
standing of  sanitary  data.  For  example,  oxygen  consumed,  nitrites, 
nitrates,  total  solids,  albuminoid  ammonia,  and  chlorides,  when 
high^  indicate  putrefactive  bacteria.  Putrefactive  bacteria  under 
proper  conditions  reduce^  often  very  strikingly,  the  oxygen  consumed, 
nitrites,  nitrates,  total  solids,  and  albuminoid  ammonia.  These 
statements  are  in  perfect  harmony.  (Septic  tank  data  would  con- 
firm this.)  Thus  as  the  organic  matter  i,s  converted  to  inorganic 
matter,  the  oxygen  consumed  (organic)  decreases  as  the  free  ammonia 
(inorganic)  increases. 

The  culture  tests,  since  they  discriminate  sharply  between  putre- 
factive changes  and  normal  variations,  seem  especially  adapted  for 
water  analysis.  The  specific  tests  for  sanitary  purposes,  which  seem 
to  be  especially  applicable,  are  free  ammonia  and  nitrites. 

In  its  present  stage  of  development  we  recognize  that,  as  in  all 
other  methods,  no  distinction  is  made  between  pollution  from  animal 
and  human  sewage.  At  times  this  distinction  may  be  of  paramount 
importance  when  we  consider  that  typhoid  bacteria  are  neither 
harbored  by  lower  animals  nor  multiply  in  natural  water. 

We  realize  that  the  foregoing  experiments  can  be  regarded  only 
as  preliminary.  Before  absolute  conclusions  can  be  drawn  more 
experimental  work  must  be  done.  We  would  suggest  the  following 
topics: 

Experiments  to  illustrate  the  action  of  pure  cultures  of  water-borne  bacteria 
on  media  of  various  composition. 

Experiments  to  determine  the  proportion  of  cultural  changes  due  to  the  bacteria 
and  to  the  chemical  condition  of  the  inoculated  water  samples. 

Experiments  with  media  of  different  composition,  with  especial  reference  to  acidity, 
and  nitrogen,  sulphur,  and  carbohydrate  compounds. 

Study  of  both  the  (i)  formation  of  putrefactive  products  and  (2)  their  fate  when 
added  to  the  original  media;  e.  g.,  the  formation  of  nitrites  from  nitrite-free  media 
rich  in  nitrates  or  saline  ammonia  and  their  removal  when  added  directly  to  the  original 


Chemical  Examination  of  Water  Bacteria 


49 


media,  or  the  formation  of  free  ammonia  from  proteids  or  its  reactions  when  added 
to  the  original  media. 

Further  comparisons  of  water  known  to  be  pure  and  of  water  known  to  be  pol- 
luted, and  artifical  pollutions  with  sewages  and  pure  cultures,  guarding  against  any 
errors  that  might  arise  from  overgrowths. 

Experiments  with  cultures  of  different  ages  and  temperatures. 

Experiments  on  the  possibility  of  mailing  small  un-iced  samples.  The  forms 
ordinarily  multiplying  under  these  conditions  are  not  the  ones  which  develop  at  37°.^ 

Put  pure  cultures  in  water,  dilute  and  incubate  with  different  dilutions. 

It  is  our  hope  that  other  investigators  in  the  field  of  water  analysis 
may  take  up  with  us  the  study  along  the  lines  suggested.  In  closely 
related  work  on  the  bio-chemistry  of  sewage  filters  Gage  and  Clark^ 
have  already  reported  considerable  success. 

In  conclusion  we  wish  to  express  our  thanks  to  Dr.  Edward  Bartow 
for  his  invaluable  co-operation  in  the  development  of  the  theoretical 
and  practical  considerations  involved  in  the  work  and  to  Mr.  P.  C. 
Jeans  and  Mr.  J.  M.  Lindgren  who  have  rendered  material  assistance 
in  carrying  on  the  analytical  work. 

* Prescott  and  Winslow,  Elements  of  Water  Bacteria,  New  York,  1904. 

® Eng.  News,  1905,  53,  p.  27,  and  Jour.  Am.  Chem.  Soc.,  1905,  27,  p.  327. 


